1. Field of the Invention
The present invention concerns a method to sequence a genomic DNA, a cloned DNA, a single stranded DNA or an oligonucleotide. One embodiment of the inventive process couples amplification of genomic DNA via the polymerase chain reaction (PCR) with DNA sequencing via dideoxynucleotide chain termination.
2. Background of the Invention
The polymerase chain reaction is described in Mullis and Faloona, 1987, Methods Enzymol., 155, 335 and in U.S. Pat. Nos. 4,683,195 and 4,683,2.02. The entire contents of U.S. Pat. Nos. 4,683,195 and 4,683,202 are incorporated by reference herein.
DNA sequencing via dideoxynucleotide chain termination is described in Sanger et al, 1977, Proc. Natl. Acad. Sci. USA, 74, 5463.
Direct sequencing of PCR products has proven difficult because of reannealing of the complementary strands, which often disturbs primer extension along the template. Although asymmetric PCR and M13 cloning of PCR products produce single stranded templates suitable for sequencing, these methods require multiple preparative steps.
Many prior protocols require nested primers for the sequencing steps.
Asymmetric PCR (Gyllenstein and Erlich, 1988, Proc. Natl. Acad. Sci. USA, 85, 7652) is a procedure for sequencing PCR products. Asymmetric PCR generates an excess of one DNA strand which is to be sequenced. Considerable empirical testing for every template is required to obtain sequence data from both strands for confirmation.
Most heretofore sequencing methods rely on a single primer extension reaction With chain-terminating dideoxynucleotides.